Monoclonal antibodies specific for toxin b of clostridium difficile

ABSTRACT

Monoclonal antibodies specific for Toxin B of Clostridium difficile are provided. Further, methods for making and using the antibodies are given, particularly the use of the antibodies for the detection of C. difficile.

BACKGROUND OF THE INVENTION

Clostridium difficile is the major cause (95%) of disease in patientssuffering from antibiotic-associated pseudomembranous colitis and ismoderately associated (20%) with patients having antibiotic-associateddiarrhea without colitis (Bartlett, J., Clin Gastroenterol. 8:783-801(1979)). In addition, 19% of patients with chronic inflammatory boweldisease have the C. difficile toxin in their stools and a positivecorrelation exists between the severity of the illness and the presenceof toxin (Trnka, Y., et al., Gastroenterology 80:693-696 (1981)).

There are two toxins, Toxin A and Toxin B, produced by C. difficile(Sullivan, N. M., Infect. Immun. 33:1032-1040 (1982)). Toxin A, orenterotoxin, is responsible for the increase in intestinal permeabilityassociated with disease (Triadafilopoulos, G., et al., Gastroenterology93:273-279 (1987)). Toxin B, or cytotoxin, is a thousand-fold morepotent than Toxin A in triggering the cytotoxic effect in cultured cells(Rothman, S. W., Infect. Immun. 46:324-331 (1984); Sullivan, N. M.,Infect. Immun. 33:1032-1040 (1982)).

Toxin A has been reported to have a molecular weight greater than300,000 kDa while Toxin B is slightly smaller (Lyerly, D. H., et al.,Infect. Immun. 54:70-76 (1986)). Further, antisera made against onetoxin is not cross-reactive with the other. Thus, the two toxins havedistinct biological and serological activity.

Although specific antibiotic therapy exists, a rapid and accuratediagnostic assay for the toxins responsible for the disease does notexist. A rapid latex test has been developed, however, it detects a43,000 molecular weight C. difficile associated protein (Lyerly, D. M.,et al., J. Clin. Microbiol. 26:397-400 (1988)) that is only weakly (67%)associated with clinically defined disease (Peterson, L. R., Am. J.Clin. Path. 87:298-299 (1987)). The in vitro cell cytotoxicity assay isa widely accepted diagnostic for C. difficile associated disease.Unfortunately, this test requires 48 hours to perform and requirestechnicians skilled in tissue culture. The performance of toxin specificpolyclonal based EIA have been disappointing. This is because of therelatively low toxin specific titer and the high level of nonspecificreactivity of the polyclonal antisera (Walter, R. C., et al.. Diagn.Microbiol. Infect. Dis. 5:61-69 (1986)).

Despite their potential utility in the detection of disease, thegeneration of toxin-specific hybridomas has proven difficult. Lyerly etal., supra. report the production of Toxin A specific monoclonalantibodies. Also, Wilkins et al. in U.S. Pat. No. 4,879,218, issued Nov.7, 1989, describe the production of Toxin A specific monoclonalantibodies. This patent also describes the production of mono-specificToxin B antibodies made from polyclonal serum containing both Toxin Aand Toxin B antibodies.

A need continues to exist for Toxin B monoclonal antibodies, withoutcross reactivity to Toxin A of C. difficile.

SUMMARY OF THE INVENTION

The present invention is drawn to antibodies, particularly monoclonalantibodies specific for Toxin B of Clostridium difficile. The antibodiesshow no reactivity to Toxin A from C. difficile. The antibodies areuseful in methods for the detection and treatment of Clostridium.

DESCRIPTION OF THE FIGURES

FIG. 1A AND 1B: Purification of Toxin B by gel permeation and anionexchange chromatography. A) Concentrated C. difficile 10463 cell culturesupernatant fractionated on a 5-300 column. B) Cytotoxin rich pooledfractions from the 5-300 column eluted from a DEAE column.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Compositions and Methods forthe Detection and Treatment of Enterocolitis Caused by C. difficile

Monoclonal antibodies are provided which are specific for Toxin B of C.difficile. The antibodies find use in assays for the detection of C.difficile and in therapies for the treatment of enterocolitis.

The monoclonal antibodies of the present invention are specific forToxin B of C. difficile. The antibodies react with a 250 kDa protein andthe supernatants of toxigenic C. difficile and are unreactive withnontoxigenic C. difficile strains. The monoclonal antibodies do notreact with concentrated supernatants from C. sordellii, C. bifermentans,C. perfingens, or the purified Toxin A from C. difficile. This is indirect contrast to the results which have been demonstrated utilizingpolyclonal antisera developed against Toxin B. Polyclonal antiseradeveloped against Toxin B reacted strongly with supernatants from C.sordellii, C. bifermentans and the nontoxigenic strain 2037.

As is generally known in the art, antibodies are deemed to be crossreactive when it binds with an antigen other than the one used to elicitformation of that antibody. By the present invention, the antibodiesagainst Toxin B do not cross react with Toxin A.

The antibodies of the present invention are prepared by immunization ofan animal, such as a rabbit, with inactive Toxin B antigens. A specificmethod 1s set forth in the experimental section which provides for therecovery of Toxin B rich fractions.

After purification of the toxin, Toxin B can be inactivated utilizingseveral methods. For example, the toxin can be inactivated utilizing SDSor alternatively by utilizing formaldehyde. Both these methods are setforth in more detail in the experimental sections. The inactivatedtoxins may be utilized to immunize animals for the production ofmonoclonal antibodies.

It is of particular importance that the monoclonal antibodies of thepresent invention do not cross react with Toxin A. While it can only behypothesized why the specific protocol of the present invention iscapable of preparing monoclonal antibodies specific for Toxin B, it isrecognized that this capability may be specific to a single aspect ofthe experimental scheme. For example, it is possible that thepurification methods set forth in the experimental section provideshighly purified Toxin B which could be utilized in other methodologiesfor the production of monoclonal antibodies. Alternatively, theinactivation means provided in the present application may have providedinactivated Toxin B with less cytotoxic effects such that the stimulatedB cells were capable of producing antibodies. It is in recognition thatthe highly purified inactivated Toxin B of the present invention iscapable of producing monoclonal antibodies that a general description ofmonoclonal antibody production is provided.

For the most part, various methodologies are well known in the art ofimmunology for the production of monoclonal antibodies. Standardreference works setting forth the general principles of immunologyinclude the work of Klein, J. (Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, New York (1982)); Kennett, R., et al.(Monoclonal Antibodies, Hybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, New York (1980)); Campbell, A. ("MonoclonalAntibody Technology," In: Laboratory Techniques in Biochemistry andMolecular Biology, Volume 13 (Burdon, R., et al., eds.), Elsevier,Amsterdam (1984)); and Eisen, H. N., (In: Microbiology, 3rd Ed. (Davis,B. D., et al., Harper & Row, Philadelphia (1980)).

The monoclonal antibodies of the invention may be "humanized" (i.e.non-immunogenic in a human) by recombinant or other technology.Humanized antibodies may be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R. etal., International Patent Publication PCT/US86/02269; Akira, K. et al.,European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison, S. L. et al., European Patent Application173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly,S. et al.. European Patent Application 125,023; Better, M. et al.,Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci.USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526(1987); Sun, L. K. et al Proc. Natl. Acad. Sci. USA 84:214-218 (1987);Nishimura, Y. et al., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al.,Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst. 801553-1559 (1988).

General reviews of "humanized" chimeric antibodies are provided byMorrison, S. L. (Science. 229:1202-1207 (1985)) and by Oi, V. T. et al.,BioTechniques 4:214 (1986)).

Suitable "humanized" antibodies can be alternatively produced by CDR orCEA substitution (Jones, P. T. et al., Nature 321:552-525 (1986);Verhoeyan et al., Science 239:1534 (1988); Beidler, C. B. et al., J.Immunol. 141:4053-4060 (1988)).

An antibody is said to be "capable of binding" a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The specific reaction referred to above ismeant to indicate that the antigen will react, in a highly selectivemanner, with its corresponding antibody and not with the multitude ofother antibodies which may be evoked by other antigens.

The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used hereinis meant to include intact molecules as well as fragments thereof (suchas, for example, Fab and F(ab')₂ fragments) which are capable of bindinghapten. Fab and F(ab')₂ fragments lack the Fc fragment of intactantibody, clear more rapidly from the ciruclation, and may have lessnon-specific tissue binding of an intact antibody (Wahl et at., J. Nucl.Med. 24:316-325 (1983)). It will be appreciated that Fab and F(ab')₂ andother fragments of the antibody of the present invention may be usedaccording to the methods of the present invention for the detection andtreatment of colon adenocarcinoma in the same manner as intact antibody.Such fragments are typically produced by proteolytic cleavage, such aspapain (to produce Fab fragments) or pepsin (to produce F(ab')₂fragments). Alternatively, hapten-binding fragments can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry.

Monoclonal antibodies are prepared using hybridoma technology (Kohler etal., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al.,In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.563-681 (1981)). In general, such procedures involve immunizing ananimal with inactive purified Toxin B. The splenocytes of such animalare extracted and fused with a suitable myeloma cell line. Any suitablemyeloma cell line may be employed in accordance with the presentinvention; however, it is preferable to employ the parent myeloma cellline (SP₂ O), available from the American Type Culture Collection,Rockville, Md. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described Wands, J.R., et al., Gastroenterology 80:225-232(1981), which reference is herein incorporated by reference). Thehybridoma cells obtained through this selection are then assayed toidentify clones which secrete antibodies specific for Toxin B. Thepreferred hybridoma cell lines are 5 AB and 5A2.

Through application of the above-described methods, additional celllines capable of producing antibodies specific for Toxin B can beobtained.

Alternatively, additional antibodies may be produced in a two-stepprocedure through the use of anti-idiotypic antibodies. Such a methodmakes use of the fact that antibodies are themselves haptens, and that,therefore, it is possible to obtain an antibody which binds to a secondantibody. In accordance with this method, antibodies capable of bindingToxin B are used to immunize an animal. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce antibody capable ofbinding antibody to Toxin B. Such antibodies comprise anti-idiotypicantibodies. Such antibodies can be used to immunize an animal, andthereby induce the formation of anti-Toxin B antibodies. Hence,antiidiotypic antibodies provide one method for inducing, or enhancing,an animal's immune response.

The antibodies (or fragments thereof) of the present invention areparticularly suited for use in immunoassays wherein they may be utilizedin liquid phase or bound to a solid-phase carrier.

Antibodies, or fragments thereof, may be labeled using any of a varietyof labels and methods of labeling. Examples of types of labels which canbe used in the present invention include, but are not limited to, enzymelabels, radioisotopic labels, non-radioactive isotopic labels,fluorescent labels, toxin labels, and chemiluminescent labels.

Examples of suitable enzyme labels include malate hydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, betagalactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,etc.

Examples of suitable radioisotopic labels include ³ H, ¹²⁵ I, ¹³¹ I, ³²P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ⁵⁷ To, ⁵⁸ Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu,²¹⁷ Ci , ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, and ¹⁰⁹ Pd.

Examples of suitable fluorescent labels include an ¹⁵² Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, a fluorescamine label, etc.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin. Examples of chemiluminescent labels include a luminallabel, an isoluminal label, an aromatic acridinium ester label, animidazole label, an acridinium salt label, an oxalate ester label, aluciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedy,J. H., et al. (Clin. Chim. Acta 70:1-31 (1976)), and Schurs, A. H. W.M., et al. (Clin. Chim. Acta 81:1-40 (1977)). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all these methodsincorporated by reference herein.

The detection of the antibodies (or fragments of antibodies) of thepresent invention can be improved through the use of carriers.Well-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. Those skilled in the art will note many othersuitable carriers for binding monoclonal antibody, or will be able toascertain the same by use of routine experimentation.

The antibodies, or fragments of antibodies, of the present invention maybe used to quantitatively or qualitatively detect the presence of ToxinB antigen. Such detection may be accomplished using any of a variety ofimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect SF-25 antigen through theuse of radioimmune assays. A good description of a radioimmune assay(RIA) may be found in laboratory techniques and biochemistry inmolecular biology, by Work, T. S., et al., North Holland PublishingCompany, NY (1978), with particular reference to the chapter entitled"An Introduction to Radioimmune Assay and Related Techniques" by Chard,T., incorporated by reference herein.

The binding molecules of the present invention may also be adapted forutilization in an immunometric assay, also known as a "two-site" or"sandwich" assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportthat is insoluble in the fluid being tested (i.e., blood, lymph,liquified stools, tissue homogenate, etc.) and the quantity of solubleantibody bearing the label that permits detection and/or quantitation ofthe ternary complex formed between solid-phase antibody, antigen, andlabeled antibody is added.

Typical immunometric assays include "forward" assays in which theantibody bound to the solid phase is first contacted with the samplebeing tested to extract the antigen from the sample by formation of abinary solid phase antibody-antigen complex. After a suitable incubationperiod, the solid support is washed to remove the residue of the fluidsample, including unreacted antigen, if any, and then contacted with thesolution containing an unknown quantity of labeled antibody. After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support through the unlabeled antibody,the solid support is washed a second time to remove the unreactedlabeled antibody. This type of forward sandwich assay may be a simple"yes/no" assay to determine whether antigen is present or may be madequantitative by comparing the measure of labeled antibody with thatobtained for a standard sample containing known quantities of antigen.These "two-site" or "sandwich" assays are described by Wide at pages199-206 of Radioimmune Assay Method. edited by Kirkham and Hunter, E. &S. Livingstone, Edinburgh, 1970.

In another type of "sandwich" assay, which may also be useful with theantigens of the present invention, the so-called "simultaneous" and"reverse" assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support and labeledantibody are both added to the sample being tested at the same time.After the incubation is completed, the solid support is washed to removethe residue of fluid sample and uncomplex labeled antibody. The presenceof labeled antibody associated with the solid support is then determinedas it would be in a conventional "forward" sandwich assay.

In the reverse assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the simultaneous and forward assays.

As explained above, the immunometric assays for antigen require that theparticular binding molecule be labeled with a "reporter molecule." Thesereporter molecules or labels, as identified above, are conventional andwell-known to the art. In the practice of the present invention, enzymelabels are a preferred embodiment. No single enzyme is ideal for use asa label in every conceivable immunometric assay. Instead, one mustdetermine which enzyme is suitable for a particular assay system.Criteria important for the choice of enzymes are turnover number of thepure enzyme (the number of substrate molecules converted to product perenzyme site per unit of time), purity of the enzyme preparation,sensitivity of detection of its product, ease and speed of detection ofthe enzyme reaction, absence of interfering factors or of enzyme-likeactivity in the test fluid, stability of the enzyme and its conjugate,availability and cost of the enzyme and its conjugate, and the like.Included among the enzymes used as preferred labels in the immunometricassays of the present invention are peroxidase, alkaline phosphatase,betagalactosidase, urease, glucose oxidase, glycoamylase, malatedehydrogenase, and glucose-6-phosphate dehydrogenase. Urease is amongthe more preferred enzyme labels, particularly because of chromogenic pHindicators which make its activity readily visible to the naked eye.

As used herein, an effective amount of a diagnostic reagent (such as anantibody, antibody fragment, or a hapten) is one capable of achievingthe desired diagnostic discrimination. The amount of such materialswhich are typically used in a diagnostic test are generally between0.1-1 micron g, and preferably between 0.1-1 micron g.

In addition to providing a method for diagnosing colitis, the presentinvention also provides a means for preventing and for treatingenterocolitis.

EXPERIMENTAL

Bacterial strains and growth conditions. The toxigenic strain 10463 andthe nontoxigenic strain 2037 of C. difficile as well as C. sordellii, C.bifermentans and C. perfringens were the generous gifts of Dr. ThomasLaMont (University Hospital, Boston, Mass.). Cells were grownanaerobically in Brain Heart Infusion Broth (Scott Laboratories, Carson,Calif.) at 37° C. until stationary phase was reached.

Toxin purification. 400 ml of bacterial supernatant was eitherconcentrated 25 fold on a YM-30 membrane (Amicon Corp., Danvers, Mass.)or precipitated using 70% ammonium sulfate. The concentrated supernatantwas then fractionated on a 90×2.5 cm column of Sephacryl-300(Pharmacia-LKB, Piscataway, N.J.). The pooled toxin containing fractionswere then applied to a superfine DEAE-TSK column (Toyo Soya, Japan)attached to a Waters 650 Advanced Protein Purification System(Millipore, Bedford, Mass.). The sample was loaded in 50 mM Tris, pH 8.0and eluted in 90 mM Tris, 1M NaCl, pH 8.0. In all cases, Toxin B richcolumn fractions were identified by the cytotoxicity assay. Theefficiency of recovery as judged by cytotoxicity was 20% after anionexchange. Toxin A was purified by the method of Sullivan et al.(Sullivan, N. M., Infect. Immun. 33:1032-1040 (1982)).

Toxin B was inactivated using two methods. SDS inactivated Toxin B wasgenerated using 100 ug/ml of Toxin B in 0.5% sodium dodecyl sulphate andheating at 100° C. for 90 seconds. Formaldehyde inactivated Toxin B wasmade by incubating 1 mg/ml of ToxinB with 0.4% formaldehyde at 37° C.for 36 hours.

Immunization of animals and generation of hybridoma cell lines. A 5pound female New Zealand White rabbit, R46O, was injected intradermallyon days 0, 21, 42, 63, 90, 117 and 144 with 100 ug of formaldehydeinactivated Toxin B. The first immunization was in complete Freund'sadjuvant while all subsequent injections were in incomplete Freund'sadjuvant. Serum was collected one week after the last immunization. Twoadditional rabbits, R458 and R459, were immunized using an identicalschedule with 250 ug of formaldehyde inactivated crude Toxin B. CrudeToxin B represents material that had not been purified to nearhomogeneity by DEAE chromatography.

Female RBF/DnJ mice were obtained from Jackson Laboratories (Bar Harbor,ME). The mice were immunized on day 0 intradermally with 5 ug of SDSinactivated Toxin B in complete Freund's adjuvant, on day 19intraperitoneally with 5 ug of SDS inactivated Toxin B in incompleteFreund's adjuvant, on day 30 intraperitoneally with 5 ug of SDSinactivated Toxin B, on day 42 intraperitoneally with 2 ug SDSinactivated Toxin B, on day 55 intravenously with 2 ug of heatinactivated Toxin B, and on day 127 intraperitoneally with 1 ug of heatinactivated Toxin B, and on day 128 intravenously with 1 ug of heatinactivated Toxin B. On day 130, a fusion was done on cells derived fromlymph nodes and spleen.

Fusion screening. The hybridomas were screened for their ability tocapture Toxin B in an EIA format. Briefly, Immulon I plates (Fisher,Boston, MA) were coated with 100 ul of 2.8 ug/ml goat anti-mouse IgG(heavy and light chain) (Boehringer Mannheim Biochemicals, Indianapolis,Ind.) in water. 100 ul of 0.005% glutaraldehyde was then added and theplates were left at room temperature overnight. Plates were then washedonce with 10 mM sodium phosphate pH 7.2 with 0.1% sodium azide. Plateswere then blocked with 200 ul of 4% bovine serum albumin in 10 mM sodiumphosphate with azide overnight. Blocked plates were then overlaid with200 ul of 2.5% sucrose in 10 mM sodium phosphate with azide overnight.Plates were then air dried and stored at 4° C. in a desiccated pouch.

100 ul of tissue culture supernatant diluted 1:10 in 50 mM Tris HCl, pH7.6, 50% heat inactivated calf serum, 0.05% Tween 20, 0.02% thimerosal,and 0.0016% gentamicin was added to each well for 1 hour at roomtemperature. Plates were then washed five times with 200 ul of 0.05%Tween 20. Plates were then incubated for one hour at room temperaturewith 500 ng/ml of Toxin B in fetal calf serum diluent. Plates were thenwashed five times with 0.05% Tween 20 and then overlaid with 100 ul HRPconjugated to antisera developed against crude Toxin B. Plates were thenwashed five more times with 0.05% Tween 20 and developed with TMB/H₂ O₂.

Direct EIA. 96 well Immulon I plates were coated with 100 ul of tissueculture supernatants of either C. difficile 10463, or 2037, C.sordellii, C. bifermentans, or C. perfringens overnight at roomtemperature. Plates were then blocked with 4% bovine serum albumin inphosphate buffered saline, 0.1% sodium azide, pH 7.0 overnight at roomtemperature. 100 ul of hybridoma tissue culture supernatant diluted 1:10or rabbit sera diluted at 1:5000 in BSA-PBS was then added to each welland incubated for two hours at room temperature. Plates were then washedfive times with PBS followed by the addition of 100 ul of 1:5000dilution of HRP conjugated to either goat anti-mouse Ig (heavy and lightchain) or antirabbit (IgG F(ab')₂) in BSA-PBS for one hour. After fivemore 200 ul washes with PBS the wells were developed as before.

Miscellaneous methods. Isotyping reagents were obtained from SouthernBiotechnology Associates, Inc. (Birmingham, Ala.). Toxin containingsamples were subjected to sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). Samples were electrophoretically transferredto nitrocellulose and immunostained using either neat hybridoma tissueculture supernatants or polyclonal antisera diluted 1:1000 into BSA-PBS.Detection was accomplished using HRP conjugated to either goatanti-mouse IgG (heavy and light chain) or anti-rabbit IgG F(ab')2 withminimum cross reactivity to human serum proteins (Pel Freez, Rogers,Ark.). 4-chloro-l-naphthol was used to develop specific antibody. Acytotoxicity assay was performed on human newborn foreskin diploidfibroblasts obtained from Bartell Immunodiagnostics (Bellevue,Washington). Samples were judged to be positive for cytotoxicity after48 hours if they produced cell rounding that was inhibitable byanti-toxin sera.

RESULTS

Purification of antigens. Toxin B purification was accomplished byconcentration, size exclusion chromatography, and anion exchangechromatography. There was virtually no loss of cytotoxicity afterconcentration. FIG. 1A shows a typical fractionation of concentratedbacterial supernatant by gel chromatography. In this case, fractions 44through 66 were pooled. This step had a 60% efficiency of cytotoxinrecovery. FIG. 1B shows an elution profile of 5-300 purified materialfrom a DEAE column. Fractions 63-75 were pooled. Although Coomassie bluestained gels of DEAE purified Toxin B showed that the protein was nearlyhomogeneous, less than 10% of the initial cytotoxicity was recovered.

Hybridomas. Of 886 fusion wells plated, 502 were positive for growth,and 213 wells produced antibody which were reactive in Toxin B captureassay. Six wells were cloned and expanded for subsequent analysis.Similar fusions resulted in a much lower percentage of specific wells.Hybridomas derived from those fusions frequently produced antibodieswhich reacted with minor low molecular weight non-Toxin B contaminantsin Western blot.

Direct EIA. In a direct EIA tissue culture, supernatants from all sixmonoclonal antibodies reacted strongly with both purified Toxin B andsupernatants from the toxigenic C. difficile strain 10463 (Table 1).None of the monoclonal antibodies tested reacted with either purifiedToxin A or supernatants from C. sordellii, C. bifermentans, C.perfringens,or the nontoxigenic C. difficile strain 2037. In sharpcontrast, all of the polyclonal antisera cross reacted with C. sordelliiand bifermentans as well as the nontoxigenic C. difficile strain 2037.The degree of cross reactivity was variable and independent of antigenpurity. None of the polyclonal antisera were reactive with either ToxinA or C. perfringens. Pre-immune sera from these rabbits were unreactivewith any of the antigens tested.

                                      TABLE 1                                     __________________________________________________________________________                         c. difficile                                                        Pure C. difficile                                                                       supernatant                                                                         Supernatants from clostridium                      Mab.sup.a                                                                           Isotope                                                                            Toxin A                                                                            Toxin B                                                                            10463                                                                            2037                                                                             sordellii                                                                          bifermentans                                                                         perfringens                            __________________________________________________________________________    5A2   IgG2b,k                                                                            0.083                                                                              2.819                                                                              1.592                                                                            0.087                                                                            0.052                                                                              0.042  0.054                                  5C8   IgM,k                                                                              0.162                                                                              3.000                                                                              2.011                                                                            0.069                                                                            0.073                                                                              0.103  0.074                                  7D8   IgGl,k                                                                             0.063                                                                              1.837                                                                              0.777                                                                            0.033                                                                            0.033                                                                              0.037  0.043                                  5A8   IgGl,k                                                                             0.071                                                                              3.000                                                                              3.000                                                                            0.029                                                                            0.028                                                                              0.038  0.039                                  1D5   IgGl,k                                                                             0.131                                                                              3.000                                                                              3.000                                                                            0.062                                                                            0.052                                                                              0.064  0.173                                  1G6   IgGl,k                                                                             0.086                                                                              3.000                                                                              3.000                                                                            0.042                                                                            0.062                                                                              0.048  0.055                                  Polyclonal.sup.b                                                              R458  NR   0.057                                                                              2.108                                                                              3.000                                                                            0.977                                                                            1.077                                                                              0.966  0.137                                  R459  NR   0.039                                                                              0.885                                                                              2.348                                                                            0.348                                                                            0.329                                                                              0.372  0.093                                  R460  NR   0.050                                                                              2.457                                                                              1.362                                                                            1.752                                                                            1.247                                                                              0.908  0.079                                  __________________________________________________________________________     .sup.a Using a 1:10 dilution of the indicated monoclonal antibody tissue      culture supernatant and HRP conjugated goat antimouse Ig (heavy and light     chain) for detection as described in the Materials and Methods section.       .sup.b Using a 1:5000 dilution of rabbit polyclonal antisera and HRP          conjugated goat antirabbit IgG F(ab')2 with minimum cross reactivity to       human servm proteins (Pel Freez, Rogers, AR).                                 MR = Not relevant.                                                       

Western blot analysis of C. difficile toxin. Western blot analysis wasdone in SDS-PAGE of, S-300 purified Toxin B with R460, R459, R458, 5C8,5A2, 7D8, 1D5AB, and 1G6. Antisera was diluted 1:1000, while monoclonalantibodies were used as tissue culture supernants. All of the monoclonalantibodies and the polyclonal antisera were reactive with an identicalband in the concentrated supernatant from the toxigenic C. difficilestrain 10463 and in the purified Toxin B (results not shown). Thepolyclonal antisera also recognized major bands at 40 and 55 kDa with aminor set of triplet bands at approximately 100 kDa.

All of the Toxin B specific monoclonal antibodies also detected avariable amount of a 50 kDa protein which was present in theconcentrated cell culture supernatants of C. difficile. Western blotswere performed of Toxin B and C. sordellii following trypsin treatment.Cell culture supernatants of either C. difficile 10463 or C. sordelliiwere precipitated with 70% ammonium sulfate, resuspended in 0.1M Tris,pH 8.0, and desalted on a Sephadex G-2 5 column that had beenpre-equilibrated with 20 mM Tris-HCl, pH 8.0. Undigested C difficile orC. sordellii proteins were compared. C. difficile or C. sordelliiproteins were digested for 30 minutes at 37° C. with trypsin at a 1:50weight ratio of enzyme to bacterial protein. (Results not shown.)Although very antigenic, this material was substantially less cytotoxicthan high molecular weight Toxin B (FIG. 1A) and could be removed byS-300 chromatography. A similar set of immunoreactive bands (60 and 250kDa) could be seen in the concentrated cell culture supernatants of C.sordellii using the monoclonal antibodies 5A2 (not shown) and 5C8. Theother monoclonal antibodies were completely unreactive with C. sordelliiin Western blots. All of the immunoreactive bands were trypsinsensitive, however, they did not produce identical break down products.The lowest molecular weight immunoreactive trypsin fragment of C.difficile Toxin B was 50 kDa while the lowest molecular weight trypsinfragment of C. difficile was 79 kDa.

CONCLUSIONS

While Lyerly et al. Infect. Immun. 54:70-76 (1986) showed that one oftheir Toxin A specific monoclonal antibodies weakly cross reacted withToxin B, none of the six Toxin B specific monoclonal antibodies crossreacted with Toxin A. Furthermore, the rabbit polyclonal antiseradeveloped against purified Toxin B as disclosed herein was unreactivewith pure Toxin A. This, of course, does not preclude the existence ofconserved domains in the two toxins but does suggest that these domainsare not immunologically dominant.

Despite the rather high frequency of Toxin B specific hybridomasobserved in this fusion, previous attempts were much less successful.The cytotoxic effects of Toxin B required the extreme proceduresdescribed herein to complete inactivation. Even with this precaution,mice were generally quite sick after immunization. A possibleexplanation is that denaturation of Toxin B was sufficiently harsh thatstimulated B cells infrequently produced antibodies which recognized thenative antigen used in the fusion screening process. In addition, manywells which scored positive in the direct Toxin B screening assay laterproved to be reactive with heat stable minor proteins which contaminatethe immunogen. Presumably these are the same proteins which areprominent in the Western blots of Toxin B developed using rabbitantisera to Toxin B.

Antisera to C. sordellii lethal toxin has been reported to cross reactwith Toxin B but not Toxin A of C. difficile (Popoff, M. R., Infect.Immun. 55:35-43 (1987)). Antisera to C. sordellii toxin has even beenshown to neutralize C. difficile cytotoxicity (Chang, T. W., et al.,Infect. Immun. 22:418-422 (1978)). None of the six monoclonal antibodieswere reactive in a direct EIA with cell culture supernatant of C.sordellii. Two of the monoclonal antibodies, 5C8 and 5A2, did detect ahigh molecular weight protein in the Western blot of concentrated cellculture supernatants of C. sordellii. This protein like Toxin B wastrypsin sensitive but did not produce immunoreactive fragments with thesame molecular weight. None of the monoclonal antibodies detected thisantigen in direct EIA. It is likely that differences in the expressionlevel or the ability to coat plastic account for the lack of reactivityof any of the monoclonal antibodies with C. sordellii in direct EIA. Itis also possible that the epitope recognized by these monoclonalantibodies is only exposed on denatured C. sordellii toxin. The presentToxin B specific rabbit antisera were reactive with C. sordellii andwith C. bifermentans in direct EIA, but were unreactive with C.perfringens. Although some of this reactivity may have been due toconserved epitopes on Toxin B, other antigens in the cell culturesupernatants may also contribute to the observed reactivity. This issupported by the strong direct EIA reactivity of the rabbit antiserawith the nontoxigenic strain of C. difficile.

Results with the Toxin B specific polyclonal antisera are similar tothose previously reported. The trace contaminants seen in Coomassiestained gels dominate the reactivity of antisera to concentratedsupernatants of toxigenic C. difficile in Western blot. Thesecontaminants are thus either highly immunogenic or relatively resistantto heat and detergent denaturation. None of the monoclonal antibodiesdescribed here are reactive with these bands and it is thereforeunlikely that the lower molecular weight fragments are subunit productsof high molecular weight Toxin B. It is the high level of reactivitywith antigens other than toxin and the relatively low toxin specifictiters of the Toxin B antibodies that have hindered the development of areliable C. difficile diagnostic.

Preliminary evidence shows that these monoclonal antibodies will beuseful in the detection of Toxin B in the stools of patients with C.difficile associated disease.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

DEPOSIT INFORMATION

Cell lines 5A8 and 5A2 were deposited with the American Type CultureCollection, Rockville, Md. on May 11, 1990 and given the designationATCC No. HB10455 and ATCC No. HB10454, respectively.

What is claimed is:
 1. A monoclonal antibody specific for Toxin B ofClostridium difficile which is not cross-reactive with Toxin A ofClostridium difficile.
 2. The monoclonal antibody of claim 1 wherein themonoclonal antibody is produced from a hybridoma selected from the groupconsisting of 5C8, 5A2, 7D8, 5A8, and 1D5.
 3. A hybridoma which producesa monoclonal antibody specific for Toxin B of Clostridium difficile,wherein said monoclonal antibody is not cross-reactive with Toxin A ofClostridium difficile.
 4. The hybridoma of claim 3 wherein saidhybridoma is selected from the group consisting of 5C8, 5A2, 7D8, 5A8,and 1D5.
 5. A method for detecting the presence of Clostridium difficileToxin B in a sample comprising contacting said sample with a monoclonalantibody specific for Clostridium difficile Toxin B; anddetecting thebinding of said antibody to Clostridium difficile Toxin B.
 6. The methodaccording to claim 5, wherein said antibody is labeled.
 7. The methodaccording to claim 5, wherein said antibody is attached to a solidsupport.
 8. The method according to claim 5, wherein said sample is abiological sample.
 9. The method according to claim 8, wherein saidsample is a stool sample.